The abundant hexameric AAA ATPase, P97/VCP/Cdc48, participates in a wide range of cellular processes and is directed into these different pathways through its interaction with a large number of adaptors and cofactors. One group of adaptors consists of proteins that contain a UBX domain. There are at least 13- UBX domain-containing proteins encoded in the human genome, five of which also contain a UBA domain and interact with polyubiquitin conjugates. P97 has been proposed to function as a "separase", disassembling protein complexes through the application of mechanical force generated by the hydrolysis of ATP. However, studying the mechanism of p97 function in detail has remained difficult because for almost all the UBX adaptors soluble substrates have not yet been discovered. Recently the Deshaies laboratory identified Hypoxia-inducible factor 1 alpha (HIF-1 a) as the first soluble substrate whose efficient degradation required p97 and UBXD7. In addition, UBXD7 was found in association with a variety of ubiquitin E3 ligases including the HIF-1 a E3 ligase, Cullin2/VHL. The focus of this proposal is to use HIF-1a degradation as a model system to study the role of the p97/UBX complex in ubiquitin dependent degradation. This work will address the following questions: Aim#1 What determines the association between UBXD7 and Cullin2/VHL? Using a variety of biochemical techniques we will determine whether UBXD7-Cullin2 association depends on E3 ligase activity, or is mediated by a direct physical interaction independent of substrate ubiquitination. Aim#2 Which pool of HIF-1 a is regulated by p97? This aim will first address the relative abundance of the various HIF-1 a complexes in cells (monomer vs free heterodimer bound to HlF-1beta vs DNA-bound heterodimer) and then determine which one is regulated by p97. In Aim#3 we will reconstitute HIF-1a ubiquitination and degradation in a cell free system and, by using extracts in which specific components are depleted and/or added back, we will study the role of p97 and UBXD7. And lastly, in Aim#4 we will describe the development of a two-step purification approach followed by mass spectrometry analysis designed to identify additional substrates for UBXD7. Selective protein degradation plays an important role in many biological processes and its disruption frequently contributes to the development of neurological disorders and malignancies such as cancer. Given that its adaptors interact with numerous ubiquitin E3 ligases and bind polyubiquitin conjugates, it is likely that p97 functions as a central player in the degradation of many clinically important substrates. Therefore characterization of p97 functions and identification of novel substrates will ultimately help pave the way for the design of selective therapeutics.